Benaroya Research Institute, Seattle, Washington, United States of America.
PLoS One. 2013;8(1):e53202. doi: 10.1371/journal.pone.0053202. Epub 2013 Jan 21.
Secreted protein, acidic and rich in cysteine (SPARC) has been described as a counteradhesive matricellular protein with a diversity of biological functions associated with morphogenesis, remodeling, cellular migration, and proliferation. We have produced mouse SPARC with a FLAG-tag at the N-terminus of SPARC (Flag-SPARC, FSP) in a Bac-to-Bac baculoviral expression system. After affinity purification, this procedure yields SPARC of high purity, with an electrophoretic mobility of ∼44 kDa under reducing conditions, and ∼38-39 kDa under non-reducing conditions. Unexpectedly, FSP adsorbed to plastic supported cell attachment and spreading, in a calcium-dependent manner. The adhesive activity of native FSP was inhibited by prior incubation with anti-SPARC IgG. Cell adhesion to FSP induced the formation of filopodia and lamellipodia but not focal adhesions that were prominent on cells that were attached to fibronectin. In addition, FSP induced the tyrosine phosphorylation of FAK and paxillin in attached epithelial cells. Erk1/2 and Rac were also activated in cells attached to FSP, but at a lower level in comparison to cells on fibronectin. This study provides new insight into the biological functions of SPARC, a matricellular protein with important roles in cell-extracellualr matrix interactions.
富含半胱氨酸的酸性分泌蛋白（SPARC）被描述为一种具有多种生物学功能的抗黏附基质细胞外基质蛋白，与形态发生、重塑、细胞迁移和增殖有关。我们在 Bac-to-Bac 杆状病毒表达系统中在 SPARC 的 N 端产生了带有 FLAG 标签的小鼠 SPARC（Flag-SPARC，FSP）。经过亲和纯化，该程序可产生高纯度的 SPARC，在还原条件下电泳迁移率约为 44 kDa，在非还原条件下约为 38-39 kDa。出乎意料的是，FSP 以钙依赖性方式吸附到塑料支持的细胞附着和铺展上。天然 FSP 的粘附活性可被预先用抗 SPARC IgG 孵育所抑制。细胞黏附到 FSP 诱导形成丝状伪足和片状伪足，但不会形成在黏附到纤维连接蛋白的细胞上明显的焦点黏附。此外，FSP 诱导附着上皮细胞中 FAK 和桩蛋白的酪氨酸磷酸化。细胞附着到 FSP 后，Erk1/2 和 Rac 也被激活，但与黏附到纤维连接蛋白的细胞相比，激活水平较低。这项研究为 SPARC 的生物学功能提供了新的见解，SPARC 是一种细胞外基质相互作用中具有重要作用的基质细胞外基质蛋白。
